key: cord-0706749-cjifnqz7
authors: Du, Ruikun; Cooper, Laura; Chen, Zinuo; Lee, Hyun; Rong, Lijun; Cui, Qinghua
title: Discovery of Chebulagic Acid and Punicalagin as Novel Allosteric Inhibitors of SARS-CoV-2 3CL(pro)
date: 2021-04-17
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2021.105075
sha: 20c26f4618cb17ca6f0a70966e14d1ed944fa018
doc_id: 706749
cord_uid: cjifnqz7

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CL(pro)) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the virus that causes a pneumonia like illness pandemic called coronavirus disease 2019 . The disease has reached to almost every country in the world, posing a serious threat to global public health and the economy (Hamid et al., 2020) (Callaway, 2021; Tegally et al., 2020) . On the other hand, there is no specific antiviral therapy available for clinical usage, except remdesivir, of which the therapeutic effect is still under debate (Beigel et al., 2020) . There is a pressing need to discover additional effective antivirals for the treatment of SARS-CoV-2 infection for use alone or in combination with already approved antiviral therapies.

Broad spectrum antiviral agents are valuable candidates of inhibitors against emerging viruses .

To our knowledge, two hydrolyzable polyphenolics, chebulagic acid (CHLA, Fig. 1A ) and punicalagin (PUG, Fig.   1B ), have been well acknowledged as cost-effective and broad-spectrum antivirals. Previous studies have demonstrated CHLA and PUG can block interactions between cell surface glycosaminoglycan (GAG) and viral glycoproteins. Through this mechanism, they inhibit infections by diverse viruses that employ GAGs for host cell entry, such as herpes simplex virus type 1, human cytomegalovirus, and hepatitis C virus (Lin et al., 2011; Lin et al., 2013) . Moreover, we recently identified that CHLA and PUG possess inhibitory effects against influenza viruses by targeting neuraminidase mediated virus release Li et al., 2020c) .

In this study, we investigated the potent antiviral activity of CHLA and PUG against SARS-CoV-2.

The 

A lentiviral-based pseudovirus carrying the SARS-CoV-2 S protein (SARS-CoV-2pp) was prepared as previously described (Xia et al., 2020) , while 293T cells transiently expressing human ACE2 and TMPRSS2 were adopted as target cells (Fig. S1A ). The pseudovirus entry assay was conducted by inoculation of SARS-CoV-2pp to target cells in presence of increasing concentrations of test compound, with final concentrations ranged from 100 μM to 1.56 μM.

After incubation for 48 hours, luciferase activity was analyzed to monitor viral entry efficacy.

The SARS-CoV-2 3CL pro was prokaryotic expressed and purified as previously described with slight modification (Ma et al., 2020b) . For enzymatic inhibition assay, the recombinant 3CL pro (250 nM at a final concentration) was incubated with increasing concentrations of each compound in 90 μL reaction buffer (50 mM Tris-HCl, pH 7.3, 1 mM EDTA) and incubated for 30 min (Dai et al., 2020) . The reaction was initiated by adding 10 μL FRET-based peptidic J o u r n a l P r e -p r o o f substrate (Dabcyl-KTSAVLQ/SGFRKME-Edans) with a final concentration of 50 μM. The fluorescence signal was immediately measured every 20 s for 30 min with a Bio-Tek Synergy4 plate reader with filters for excitation at 336/20 nm and emission at 490/20 nm. The initial reaction velocities (V 0 ) of reactions were calculated to indicate the enzymatic activities. Three independent experiments were performed and IC50 curves were analyzed using GraphPad Prism software.

For the molecular docking simulations, the 3CL pro structure from SARS-CoV-2 (PDB code: 6m2n) was used. The three-dimensional (3D) structure of CHLA and PUG was obtained from PubChem Compound database (NCBI).

Before docking, polar hydrogen atoms were added to the 3CL pro protein using autodock tools. Docking was performed using a grid box covering the entire structure by AutoDock Vina software (Trott and Olson, 2010) . After docking, the conformation of the compound was analyzed, and the 3D models were viewed with PyMOL.

In order to determine whether CHLA and PUG can inhibit replication of SARS-CoV-2, a plaque reduction assay was performed with authentic SARS-CoV-2 using remdesivir as positive control. As a result, both CHLA and PUG inhibited the plaque formation of SARS-CoV-2 in a dose-dependent manner ( Fig. 2A) , with EC 50 values of 9.76 ± 0.42 μM and 7.20 ± 1.08 μM, respectively (Fig. 2B) . To exclude the possibility that the inhibition of viral replication was due to compound-mediated cytotoxicity, a cell proliferation-based cytotoxicity assay was performed. As shown in Fig glycoproteins, we therefore first tested whether CHLA and PUG act by blocking SARS-CoV-2 entry using a pseudotyped SARS-CoV-2 (SARS-CoV-2pp). However, upon adding CHLA or PUG during infection of J o u r n a l P r e -p r o o f SARS-CoV-2pp into target 293T cells transiently expressing ACE2 and TMPRSS2 (Fig. 3A) , neither CHLA nor PUG inhibited pseudovirus entry without interfering with target cell viability, indicating that CHLA and PUG inhibit SARS-CoV-2 replication by a mechanism other than preventing S-mediated viral entry (Fig. 3B ).

The 3-chymotrypsin-like cysteine protease (3CL pro ) enzyme is one of the best characterized drug targets among coronaviruses . Along with the papain-like protease (PL pro ), 3CL pro is an essential viral protease that processes the viral polyproteins, therefore, inhibiting the activity of this enzyme would block viral replication (Dai et al., 2020) . Interestingly, an in silico study revealed that several hydrolyzable polyphenolics including CHLA are potential inhibitors of the SARS-CoV-2 3CL pro (Khalifa et al., 2020) . To address this, CHLA and PUG were subjected to a SARS-CoV-2 3CL pro enzymatic inhibition assay. Upon mixture of 3CL pro with a FRET-based peptidic substrate in reaction buffer, the substrate gets hydrolyzed. After hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, resulting in an increase of fluorescence signal with proper filters (Fig.   S1A ). Both CHLA and PUG dose-dependently reduced fluorescence production (Fig. S1B) , with IC 50 values of 9.09 ± 0.87μM and 4.62 ± 0.27μM, respectively (Fig. 4) . A fluorescent interference assay further illustrated that CHLA and PUG affected fluorescence only at higher concentrations (>50 μM, data not shown). Taken together, our study revealed that CHLA and PUG can block the enzymatic activity of 3CL pro . Of note, CHLA showed little effect on the enzymatic activity of SARS-CoV-2 PL pro , while PUG slightly inhibits the PL pro activity with IC 50 of over 50 μM (Fig.   S2 ). These data suggest that PUG is a potential dual-target SARS-CoV-2 inhibitor, although its inhibitory effect against the PL pro is weak.

To better understand the kinetic modes involved in the interaction of CHLA and PUG with SARS-CoV-2 3CL pro , the proteolytic activity of the enzyme was measured using a series of enzyme concentrations and at various inhibitor concentrations. The substrate concentrations were held constant at sub-saturation levels. At each concentration of test inhibitor, the initial reaction velocities were plotted with the concentrations of 3CL pro , and fitted to a line using linear regression analysis. As indicated by Fig. 5A , as the concentration of either CHLA or PUG increases, the slope of the line decreases, suggesting that both CHLA and PUG are reversible inhibitors. In order to deduce the mode of binding, Lineweaver-Burk plots were carried out at a constant concentration of 3CL pro (250nM), increasing J o u r n a l P r e -p r o o f concentrations of substrate, and in absence or presence of various concentrations of CHLA or PUG. As a result, both CHLA and PUG exhibited noncompetitive modes of inhibition since all lines intercepting the x-axis (Fig. 5B ).

According to above enzymatic assays, both CHLA and PUG are noncompetitive allosteric inhibitors. In order to search potential allosteric binding sites, in silico docking of CHLA and PUG to 3CL pro was thereafter performed using AutoDock Vina software. As depicted in Fig. 6 , both CHLA and PUG may interact with the cleft between domain II and domain III within 3CL pro with stable binding free energy. This binding site is located a little away from the substrate binding pocket, where His41 and Cys145 form the catalytic dyad (Dai et al., 2020) .

In the urgent campaign to develop SARS-CoV-2 therapeutics, natural products from medicinal plants have been an important source of new lead compounds exhibiting different mechanisms of action, such as shikonin (Li et al., 2020a) , salvianolic acid C , cepharanthine (Rogosnitzky et al., 2020) , lycorine , ginkgolic acid and so on (Zhang et al., 2020c) . We herein report two additional natural products, CHLA and PUG as novel SARS-CoV-2 inhibitors. By binding to SARS-CoV-2 3CL pro at a pocket other that substrate binding site, CHLA and PUG act as allosteric inhibitors in reversible, noncompetitive manner.

There are two classes of inhibitors against a cysteine protease, covalent inhibitors and non-covalent ones.

Development of covalent inhibitors is usually challenging due to their toxicity and lack of specificity resulting from covalent modification of untargeted cysteine residues, while encouraging strategies have been explored, for example, by rational design, to inhibit 3CL pro via its covalent inhibition (Dai et al., 2020; Jin et al., 2020; Qiao et al., 2021) .

On the other hand, a non-covalent inhibitor may specifically bind to the target proteins reversibly. Compared to covalent inhibitors, non-covalent inhibitors can often be optimized to diffuse through membranes and bind to target proteins with high affinity (Zhang et al., 2015) . Moreover, non-covalent inhibitors are often more chemical stable than their covalent counterparts since they don't have reactive warheads，and undesirable toxic effects can be reduced for their binding to host proteins and nucleic acids are reversible (El-Baba et al., 2020) .

Virtual screens have placed hundreds chemically diverse molecules in the active site of 3CL pro , which is the main target for a non-covalent inhibitor (Douangamath et al., 2020; Gyebi et al., 2020; Khalifa et al., 2020) . Although J o u r n a l P r e -p r o o f most of the predicted inhibitors either were not subjected to antiviral assays or failed to inhibit SARS-CoV-2 replication, compound ML188 has been validated as SARS-CoV-2 inhibitor by competitively binding to 3CL pro with natural substrate (Lockbaum et al., 2021) . Besides, a non-covalent inhibitor may also act by binding to allosteric sites, which are away from the active site (El-Baba et al., 2020) . Recently, a highly reactive pocket in the dimerization region at the domain III apex of SARS-CoV-2 3CL pro has been recognized as a possible allosteric site.

Interestingly, our in silico docking data clearly reveal that both CHLA and PUG accommodates this pocket (Fig. 6) .

Historically, in order to discover non-covalent cysteine protease inhibitors, reducing reagents were usually added in the enzymatic assay of cysteine protease to prevent nonspecific covalent modification of the catalytic cysteine (Ma et al., 2020a; Ratia et al., 2008) . However, in the case of CHLA and PUG, addition of 4 mM DTT in the enzymatic assay of SARS-CoV-2 3CL pro shifted the IC 50 curves to right by 7 to 10 folds, suggesting significant loss of inhibitory effects (Fig. S3 ). This is inconsistent with our finding that CHLA and PUG are reversible, noncompetitive inhibitors of 3CL pro . To address this discrepancy, we suspect that reducing reagents might induce slight conformational changes of 3CL pro , which would not affect the detectable enzymatic activity, while the cavities where an allosteric inhibitor binds might be impaired. Although more evidence is required to support this hypothesis, the scientific community should be cautious to add reducing reagents in the enzymatic assay of cysteine protease if allosteric inhibitors were anticipated.

In summary, our study demonstrates CHLA and PUG as a novel class of SARS-CoV-2 inhibitors, by allosterically regulating the 3CL pro activity. Lineweaver-Burk plot for inhibition of CHLA or PUG on SARS-CoV-2 3CLpro. 

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We thank Dr. Shibo Jiang and Dr. Lu Lu from Fudan University for kindly providing the plasmid expressing SARS-CoV-2 Spike glycoprotein. We thank Ms. Yanyan Wang for the docking analysis. This work was supported by (1) 

The authors declare no conflict of interest.